Bifunctional antibiotics

ABSTRACT

Bifunctional antibiotics that target both bacterial RNA and resistance-causing enzymes are disclosed. The A-site of bacterial 16S rRNA serves as the target site for most aminoglycoside antibiotics. Resistance to this class of antibiotics is frequently developed by microbial enzymatic acetylation, phosphorylation or ribosylation of aminoglycosides, modifications that weaken their interactions with the target RNA. Using surface plasmon resonance (SPR), the binding affinity and stoichiometry of various amino-glycosides have been investigated and it was found that neamine, the key pharmacophore of the deoxystreptamine class of amino-glycosides, binds to the A-site in a two to one stoichiometry with a K d  of 10 μM for each binding site. A library of neamine dimers was prepared and their affinities to 16S rRNA A-site were determined by SPR, with K d =40 nM for the best dimer (an ˜10 3 -fold increase in affinity). Antibiotic activities of the dimers were determined for several bacterial strains by the Kirby-Bauer method. The most active dimer, based on antibiotic activity, also showed the highest inhibition of in vitro translation (IC 50 =0.055 μM). The latter assay was developed in order to correlate the relationship between SPR-based affinity and translation inhibition. By these combined methods, transport limitations for the semisynthetic aminoglycosides as well as non-ribosomally based antibiotic activity could be determined. Further analysis of these dimers as substrates for aminoglycoside modifying-enzymes identified a neamine dimer that was a potent inhibitor (K is =0.1 μM) of the APH(2″) activity of the bifunctional enzyme AAC(6″)-APH(2″), the primary enzyme responsible for high level gentamicin C resistance in several bacterial strains.

TECHNICAL FIELD

[0001] The invention relates to bifunctional antibiotics. Moreparticularly, the invention related to bifunctional antibiotics thattarget bacterial rRNA and inhibit resistance-causing enzymes.

BACKGROUND

[0002] Deoxystreptamine-based aminoglycosides are a clinically importantclass of antibiotics that are effective against a broad range ofmicroorganisms (Edson, R. S.; Terrel, C. L. Mayo Clin. Proc. 1991, 66,1158). It is believed that aminoglycosides exert their therapeuticeffect by interfering with translational fidelity during proteinsynthesis via interaction with the A-site rRNA on the 16S domain of theribosome (Moazed, D.; Noller, H. F. Nature 1987, 327, 389; Purohit, P.;Stern, S. Nature 1994, 370, 659; Formy, D.; et al. Science 1996, 274,1367). Unfortunately, the high toxicity and rapid emergence of highlevel aminoglycoside resistance have severely limited the usefulness ofthis class of antibiotics. Numerous aminoglycoside resistance mechanismshave been identified, and enzymatic acetylation, phosphorylation andribosylation are the primary causes of high level resistance in mostclinical isolates (Wright, G. D.; et al. Adv. Exp. Med. Biol. 1998, 456,27; Kondo, S.; Hotta, K. J. Infect. Chemother. 1999, 5, 1;Mingeot-Leclerco, M.-P.; et al. Antimicrob. Agents Chemother. 1999, 43,727). Of the modifying enzymes, the acetyl- and phosphotransferases (AACand APH) have been extensively studied with respect to their specificity(Wright, G. D.; et al. Adv. Exp. Med. Biol. 1998, 456, 27; Kondo, S.;Hotta, K. J. Infect. Chemother. 1999, 5, 1; Mingeot-Leclerco, M.-P.; etal. Antimicrob. Agents Chemother. 1999, 43, 727; Daigle, D. M.; et al.Chem. Biol. 1999, 6, 99; Azucena, E.; et al. J. Am. Chem. Soc. 1997,119, 2317; Patterson, J.-E.; Zervos, M. J. Rev. Infect. Dis. 1990, 12,644).

[0003] What was needed was a method to tackle the problem of antibioticresistance. What was needed was bifunctional aminoglycosides that canresist or inhibit aminoglycoside-modifying enzymes while simultaneouslytargeting ribosomal RNA.

SUMMARY

[0004] Bifunctional antibiotics are disclosed herein that target bothbacterial RNA and resistance causing enzymes. Preferred bifunctionalantibiotics are disclosed to be neamine dimers. These neamine dimersrepresent a new class of aminoglycoside antibiotics that arefunctionally simpler than previously known aminoglycosides. In additiontargeting bacterial RNA, they are also potent inhibitors of the APH(2″)activity of the bifunctional AAC(6′)-APH(2″) enzyme, one of the mostclinically significant of the aminoglycoside-modifying enzymes.

[0005] Bifunctional antibiotics that target both bacterial RNA andresistance-causing enzymes are disclosed and are demonstrated to providea method for tackling the problem of antibiotic resistance. The A-siteof bacterial 16S rRNA serves as the target site for most aminoglycosideantibiotics. Resistance to this class of antibiotics is frequentlydeveloped by microbial enzymatic acetylation, phosphorylation orribosylation of aminoglycosides, modifications that weaken theirinteractions with the target RNA. Using surface plasmon resonance (SPR),the binding affinity and stoichiometry of various aminoglycosides havebeen investigated and it was found that neamine, the key pharmacophoreof the deoxystreptamine class of aminoglycosides, binds to the A-site ina two to one stoichiometry with a K_(d) of 10 μM for each binding site.A library of neamine dimers was prepared and their affinities to 16SrRNA A-site were determined by SPR, with K_(d)=40 nM for the best dimer(an ˜10³-fold increase in affinity). Antibiotic activities of the dimerswere determined for several bacterial strains by the Kirby-Bauer method.The most active dimer, based on antibiotic activity, also showed thehighest inhibition of in vitro translation (IC₅₀=0.055 μM). The latterassay was developed in order to correlate the relationship betweenSPR-based affinity and translation inhibition. By these combinedmethods, transport limitations for the semisynthetic aminoglycosides aswell as non-ribosomally based antibiotic activity could be determined.Further analysis of these dimers as substrates for aminoglycosidemodifying-enzymes identified a neamine dimer that was a potent inhibitor(K_(is)=0.1 μM) of the APH(2″) activity of the bifunctional enzymeAAC(6″)-APH(2″), the primary enzyme responsible for high levelgentamicin C resistance in several bacterial strains.

[0006] One aspect of the invention is directed to a bifunctionalantibiotic. The bifunctional antibiotic includes a first and a secondpharmacophore and a linkage for linking the first and secondpharmacophore. The first and second pharmacophore each has a bindingaffininty for the A-site of bacterial 16S rRNA sufficient to inhibittranslation at clinically effective concentrations. The first and secondpharmacophores may be either identical to one another or different fromone another. The linkage has a length and structure for enabling thefirst and second pharmacophore to bind simultaneously to a single A-siteof bacterial 16S rRNA. In an improved embodiment of the invention, atleast one of the first and second pharmacophores is inhibitory ofAPH(2″) activity with respect to bifunctional enzyme AAC(6′)-APH(2″).The inhibitory activity is sufficient, at clinically effectiveconcentrations, to diminish deactivation of the bifunctional antibioticby the bifunctional enzyme AAC(6′)-APH(2″).

[0007] In one embodiment of this aspect of the invention, thebifunctional antibiotic is represented by the following structure:

[0008] In the above structure, Y¹ and Y² are the first and secondpharmacophore respectively and are both represented by:

[0009] R¹ and R² are each independently selected from the group ofradicals consisting of —H and —CH(Ph)CONHCH₂CO₂H. X is the linkage andis selected from the group of diradicals consisting of —(CH₂)_(n)— and—[(CH₂)₂O(CH₂)₃]₂O, where 3≦n≦12.

[0010] In a second embodiment of this aspect of the invention, thebifunctional antibiotic is represented by the following structure:

[0011] In the above structure, Y¹ and Y² are the first and secondpharmacophore respectively and are both represented by:

[0012] The stereochemistry is either (S,S) or (R,R). Preferred speciesof this embodiment include compounds represented by the followingstructures:

[0013] In a third embodiment of this aspect of the invention, thebifunctional antibiotic is represented by the following structure:

[0014] In the above structure, Y¹ and Y² are the first and secondpharmacophore respectively and are both represented by:

[0015] X is the linkage and is selected from the group of diradicalsconsisting of —(CH₂)_(n)— and —[(CH₂)₂]₂O, where 2≦n≦4. Thestereochemistry is either (S,S) or (R,R). Preferred species of thisembodiment include compound represented by the following structures:

[0016] Another embodiment of the above invention is directed to abifunctional antibiotic wherein the first and second pharmacophore areindependently selected from the group consisting of neamine, neomycin B,and gentamincin C₁.

[0017] Another aspect of the invention is directed to a process forinhibiting translation within a bacterium having 16S rRNA with anA-site, said process comprising the step of contacting the bacteriumwith a concentration of any of the bifunctional antibiotics describedabove sufficient to inhibit translation.

[0018] Another aspect of the invention is directed to a process forsimultaneously inhibiting translation and APH(2″) activity within abacterium having both 16S rRNA with an A-site and the bifunctionalenzyme AAC(6′)-APH(2″), said process comprising the step of contactingthe bacterium with a concentration of any of the bifunctionalantibiotics described above sufficient to inhibit translation andAPH(2″) activity.

BRIEF DESCRIPTION OF FIGURES

[0019]FIG. 1 illustrates the biotinylated E. coli 16S rRNA A-site(AS-wt) rRNA sequence.

[0020]FIG. 2 illustrates the mode of action of β-hydroxyamine commonlyfound in aminoglycoside antibiotics.

[0021]FIG. 3 illustrates is a graph showing a binding isotherm ofneamine binding to AS-wt (circles) and control mutants (U1406A, squares;U1485A, diamonds) for determination of dissociation constants(K_(d)=inverse slope) and binding stoichiometry (x-intercept).

[0022]FIG. 4 illustrates an energetic analysis of a bivalent neaminealong with a cartoon drawing illustrating how dimers are likely to bindto AS-wt rRNA with high affinity.

[0023]FIG. 5 illustrates a scheme that shows how the neamine dimers wereprepared from a known neamine precursor.

[0024]FIG. 6 illustrates a graph which demonstrates the relationshipbetween antibiotic activity (MIC, minimum inhibitory concentration) andtranslation inhibition (IC₅₀).

[0025]FIG. 7 illustrates the sites of enzymatic modification on neomycinB and gentamicin C₁.

[0026]FIG. 8 illustsrates a graph showing the results of surface plasmonresonance experiments on neomycin B binding to AS-wt rRNA and mutants.

[0027]FIG. 9 iillustrates a Scatchard plot for determining dissociationconstants (K_(d), inverse slope and binding stoichiometry (x-intercept)for the wild type organism.

[0028]FIG. 10 illutrates an Ugi reaction where four separate componentsare reacted to produce an amide-linked dimer.

[0029]FIG. 11 is a table giving the results of the Kirby Bauer test withknown compounds and the synthesized dimers.

[0030]FIG. 12 is a table that shows the minimum inhibitory concentration(MIC, μM) in E. Coli ATCC 25922 and in vitro translation IC₅₀.

[0031]FIG. 13 shows tables of the kinetic parameters of neamine andneamine dimers for various aminoglycoside-modifying enzymes.

DETAILED DESCRIPTION

[0032] The dissociation constant (K_(d)) and binding stoichiometry weredetermined using surface plasmon resonance (SPR) against an immobilizedrRNA sequence modeling the A-site of prokaryotic rRNA (FIGS. 1-4)(Hendrix, M.; et al. J. Am. Chem. Soc. 1997, 119, 3641; Wong, C.-H.; etal. Chem. Biol. 1998, 5, 397). The dissociation constants were obtainedfrom equilibrium binding curves through nonlinear curve fitting and werecomparable to those obtained using Scatchard analysis. We focused onneamine as it represents the simplest effective aminoglycosideantibiotic and contains the key β-hydroxyamine motif for interactionwith the phosphodiester group and the Hoogsteen face of guanine residuesin RNA (FIG. 2) (Hendrix, M.; et al. Angew. Chem., Int. Ed. Engl. 1997,36, 95). Neamine was found to bind biotinylated AS-wt in a 2:1 complexwith a K_(d) of 10 μM for each binding site (FIG. 3). Various dimers ofneamine were therefore constructed in order to identify a bivalentaminoglycoside that would bind AS-wt with high affinity (FIG. 4), and atthe same time resist and and/or inhibit the modifying enzymes due to itsunnatural structure (Some aminoglycoside dimers were preparedpreviously; however, the monomers bind the A-site stoichiometrically:see Michael; K.; et al. Bioorg. Med. Chem. 1999, 7, 1361; for vancomycindimers, see Rao, J.; Whitesides, G. H. J. Am. Chem. Soc. 1997, 119,10286; Sundram, U. N.; et al. J. Am. Chem. Soc. 1996, 118, 13107).

[0033] Neamine dimers were prepared starting from perbenzyl perazido5-O-carboxyethyineamine (Sucheck, S. J.; et al. Angew. Chem., Int. Ed.Engl. 2000, 39, 1080) (see FIG. 5), which was prepared from the5-O-allyl precursor (Greenberg, W. A.; et al. J. Am. Chem. Soc. 1999,121, 6527). Carboxyethylneamine was distributed into a Quest 210parallel synthesizer and was activated using a cyclohexylcarbodiimidebound to macroporous polystyrene resin. Two equivalents of resin, oneequivalent of acid and 0.4 equivalents of various diamine linkers wereutilized to synthesize a library of neamine dimers of variable linkerlength. The intermediate amides were isolated by filtration andwere >95% pure, as determined by NMR. The resulting dimers were firstreduced under Staudinger conditions to convert the azides to amines,which were captured from solution using the resin bound sulfonic acidscavenger MP-TsOH (Argonaut). The resin was washed and the free aminewas released from the resin by elution with 2 M NH₃ in methanol. Theresulting amines were debenzylated by hydrogenolysis in the presence of2 equivalents of acetic acid per amine. The reaction mixture wasfiltered, concentrated and purified by silica gel chromatography using8:2:4:5 NH₄OH—CHCl₃-n-BuOH—EtOH, followed by cation exchangechromatography to give the pure aminoglycosides dimers 4-13. Theamide-linked dimers could also be prepared via Ugi reactions, e.g. dimer14, starting from the same perbenzyl perazido 5-O-carboxyethylneamine.This procedure is also directly applicable to parallel synthesis andcould be used to increase the molecular diversity of the library.

[0034] The dimers with the highest affinity for AS-wt determined by SPRwere also the most potent antibiotics, as determined by theantimicrobial assays (Greenberg, W. A.; et al. J. Am. Chem. Soc. 1999,121, 6527; Phillips, I; Williams, D. In Laboratory Methods inAntimicrobial Chemotherapy; Gerrod, L., Ed.; Churchill LivingstonePress: Edinburg, 1978; pp 3-30) and by IC₅₀ of in vitro translation(Greenberg, W. A.; et al. J. Am. Chem. Soc. 1999, 121, 6527). Of thisseries, the dimers with the highest antibiotic activity, 4 and 6, showeda K_(d) of 1.1 μM and 0.8 μM on AS-wt, respectively, ten-fold greaterthan neamine. Dimers with longer linker lengths had weaker affinitiesfor AS-wt, a trend that correlated with antibiotic activity.Interestingly, all of the dimers continued to display a 2:1 bindingstoichiometry, indicating that the increase in affinity is most likelydue to an additional favorable (not dimeric) yet weak interaction withAS-wt. Antibiotic activities of dimers 4 and 6 were comparable toneamine, MIC=31 and 125 μM respectively, against the E. coli referencestrain (See supplement for antibiotic testing data).

[0035] The relatively weak antibiotic activity of these dimers led us todesign a flexible and hydrophilic linker by opening the1,2-propyloxiranes with an amine as shown in FIG. 5. The triflate of(S)-(−) and (R)-(+)-glycidol (Baldwin, J. J.; et al. J. Med. Chem. 1982,25, 931; Schlecker, R.; Thieme, P. C. Tetrahedron 1988, 44, 3289) wasused to alkylate perbenzyl perazido neamine to form epoxides 15 and 16,respectively. Epoxides 15 and 16 were heated for 16 h in a sealed tubewith excess methylamine to form 1,2-hydroxy amines 17 and 18,respectively. These hydroxy amines could then be used in an additionreaction with another equivalent of epoxide 15 or 16 to form dimers 19and 20, respectively, after deprotection. Epoxides 15 and 16 were alsoopened with 0.5 equivalents of a N,N′-methyldiamines to afford protecteddimers 21-28. N,N′-methyldiamine that were not commercially availablewere readily prepared by a one-pot synthesis via imine formation with aprimary diamine and benzaldehyde, alkylation of the intermediate imimewith dimethyl sulfate followed by hydrolysis of the alkylimine affordedN,N′-methyldiamines in high yield (Devinsky, F.; et al. Synthesis 1980,4, 303). The resulting dimers were deprotected as previously describedto afford dimers 21-28. These dimers possessed significantly increasedantibiotic activity compared to the amide-linked dimers. Antibioticactivity was greatest with the diaminobutane linker in dimer 27, whichshowed a MIC=6.25 μM against E. coli and K_(d)=40 nM (AS-wt) with 1 to 1stoichiometry (Compound 27 is also effective against other strains,including P. aeruginosa ATCC 27853, P. aeruginosa, PAO-1, S. aureus ATCC29213 and ATCC 33591-MRSA, and E. faecalis ATCC 29212 and is 3 timesmore effective than tobramycin against the tobramycin-resistant strainof P. aeruginosa from cystic fibrosis patients.).

[0036] To better understand the relationship between RNA binding andantibiotic activity, inhibition of in vitro translation of luciferasegene (Greenberg, W. A.; et al. J. Am. Chem. Soc. 1999, 121, 6527) wasmeasured as a function of MIC, FIG. 6. This analysis was used tovalidate the target and characterize potential transport limitations forthe aminoglycosides, and in vitro translation inhibition is expected tobe a better indicator of aminoglycoside selectivity for 16S rRNAcompared to binding affinity measurements with the A-site sequences(Hendrix, M.; et al. J. Am. Chem. Soc. 1997, 119, 3641; Wong, C.-H.; etal. Chem. Biol. 1998, 5, 397; Greenberg, W. A.; et al. J. Am. Chem. Soc.1999, 121, 6527). A nearly linear relationship between the IC₅₀ oftranslation inhibition and the MIC was observed. This analysis is usefulfor analyzing structure activity relationships within a similar seriesof compounds. Compounds falling below the line in FIG. 6 may suffer fromtransport limitation while compounds above the line may act via afundamentally different mode of action than compounds at or near theline.

[0037] Further study of neamime dimers 4, 6 and 27 using severalaminoglycoside-modifying enzymes revealed that the dimers were poorsubstrates for AAC(6′)-Ii and APH(3′)-IIIa, responsible for 6′- and3′-N-acetylation and O-phosphorylation, respectively (Wright, G. D.; etal. Adv. Exp. Med. Biol. 1998, 456, 27; Kondo, S.; Hotta, K. J. Infect.Chemother. 1999, 5, 1; Mingeot-Leclerco, M.-P.; et al. Antimicrob.Agents Chemother. 1999, 43, 727). In addition, dimers 4, 6 and 27 werepoor substrates for the AAC(6′) activity of the bifunctionalaminoglycoside modifying-enzyme AAC(6′)-APH(2″) (Wright, G. D.; et al.Adv. Exp. Med. Biol. 1998, 456, 27; Kondo, S.; Hotta, K. J. Infec.Chemother. 1999, 5, 1; Mingeot-Leclerco, M.-P.; et al. Antimicrob.Agents Chemother. 1999, 43, 727; Daigle, D. M.; et al. Chem. Biol. 1999,6, 99; Azucena, E.; et al. J. Am. Chem. Soc. 1997, 119, 2317; Patterson,J.-E.; Zervos, M. J. Rev. Infect. Dis. 1990, 12, 644), and notsubstrates for the APH(2″) activity of MC(6′)-APH(2″). They were in factpotent competitive inhibitors of the APH(2″) activity, K_(is)=0.8 μM fordimer 4, 0.1 μM for 6 and 0.7 μM for 27.

DETAILED DESCRIPTION OF FIGURES

[0038]FIG. 1 shows the biotinylated E. coli 16S rRNA A-site (AS-wt) rRNAsequence. It is this portion of the bacterial RNA on the 16S domain ofthe ribosome which is bound by the aminoglycosides. This interferes withtranslational fidelity during protein synthesis.

[0039]FIG. 2 shows the mode of action of β-hydroxyamine commonly foundin aminoglycoside antibiotics. The β-hydroxyamine motif interacts notonly with the phosphodiester group but also the Hoogsteen face ofguanine residues in RNA.

[0040]FIG. 3 is a graph showing a binding isotherm of neamine binding toAS-wt (circles) and control mutants (U1406A, squares; U1485A, diamonds)for determination of dissociation constants (K_(d)=inverse slope) andbinding stoichiometry (x-intercept). The binding is sequence selective.The inset in the figure is a Scatchard plot which shows the bindingstoichiometry.

[0041]FIG. 4 is an energetic analysis of a bivalent neamine along with acartoon drawing illustrating how dimers are likely to bind to AS-wt rRNAwith high affinity. Neamine units bind to AS-wt with a K_(d) Of 10 μMper binding site. Addition of the proper linker would enable theunnatural dimer to bind with much higher affinity and resist modifyingenzymes because of its unnatural structure.

[0042]FIG. 5 is a scheme that shows how the neamine dimers were preparedfrom a known neamine precursor. The starting material is perbenzylperazido 5-O-carboxyethylneamine which is prepared from the 5-O-allylprecursor. A variety of diamines were chosen to form a diamide linker tothe neamine units. Dimer 14 was synthesized using the Ugi reaction. Fourseparate components are added during this synthetic procedure. Syntheticsteps from the neamine expoxides are shown at the bottom of the scheme.Simple nucleophilic opening of the epoxide ring generates the dimersfrom a primary amine or a primary diamine precursor.

[0043]FIG. 6 is a graph which demonstrates the relationship betweenantibiotic activity (MIC, minimum inhibitory concentration) andtranslation inhibition (IC₅₀). The compounds above the line do nottarget RNA and have different modes of antibiotic action, while those tothe right of the line exhibit transport limitations. What was measuredis the inhibition of in vitro translation of the luciferase genemeasured as a function of MIC. This analysis was used to validate thetarget and characterize potential transport limitations for theaminoglycosides, and in vitro translation inhibition is expected to be abetter indicator of aminoglycoside selectivity for 16S rRNA compared tobinding affinity measurements with the A-site sequences. A nearly linearrelationship between IC₅₀ of translation inhibition and the MIC wasobserved.

[0044]FIG. 7 shows the sites of enzymatic modification on neomycin B andgentamicin C₁. N-acetylation, phosphorylation and O-ribosylation are themajor modifications catalyzed by resistance causing enzymes.

[0045]FIG. 8 is a graph showing the results of surface plasmon resonanceexperiments on neomycin B binding to AS-wt rRNA and mutants. The circlesare for the wild type organism and the squares and diamonds are for thetwo different mutants. The binding is sequence selective.

[0046]FIG. 9 is a Scatchard plot for determining dissociation constants(K_(d), inverse slope and binding stoichiometry (x-intercept) for thewild type organism. The binding is sequence selective.

[0047]FIG. 10 shows an Ugi reaction where four separate components arereacted to produce an amide linked dimer. Compound 14 was synthesized inthis reaction to give a linked dimer.5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzyl-neamine (60mg, 79 μmol), methyl isocyanoacetate (36 μL, 397 μmol), benzaldehyde (8μL, 79 μmol), and diaminododecane (8 mg, 40 μmol) were dissolved in amixture of anhydrous CH₂Cl₂/methanol (1:1, 800 μl). After stirring 48hours at ambient temperature, the reaction was diluted with ethylacetate (5 ml). It was then washed with 1 M HCl (2×5 ml), saturatedsodium bicarbonate (2×5 ml), and brine (1×5 ml). The aqueous extractswere re-extracted with ethyl acetate (2×5 ml). The combined organicextracts were dried (Na₂SO₄), filtered, and concentrated. Flashchromatography (silica gel, gradient hexane to 1:1 hexane/ethyl acetate)yielded protected neamine dimer 14 (21.7 mg, 26%).

[0048]FIG. 11 is a table giving the results of the Kirby Bauer test withknown compounds and the synthesized dimers The numbers under the teststrains are for diameters (mm) of zones of inhibition. All compoundsexcept neomycin and gentamicin were spotted at 200 nmoles/disk; neomycinwas spotted at 33 nmoles/disk (30 μg) while gentamicin was spotted at 10nmole/disk (10 μg). Surface plasmon resonance K_(d) values for dimers4-13 is also provided.

[0049]FIG. 12 is a table that shows the minimum inhibitory concentration(MIC, μM) in E. Coli ATCC 25922 and in vitro translation IC₅₀. The datafrom this table is graphed in FIG. 6 and shows the likely mechanism ofaction for the antibiotics.

[0050]FIG. 13 shows tables of the kinetic parameters of neamine andneamine dimers for various aminoglycoside-modifying enzymes. BF refersto the bifunctional enzyme AAC(6′)-APH(2″), where the particularactivity is indicated. The neamine data were obtained from Daigle, D.M.; et al. Chem. Biol. 1999, 6, 99.

[0051] Experimental Section:

[0052] Reactions were performed under inert atmosphere unless otherwisestated. THF and CH₂Cl₂ were distilled under Ar with benzophenone ketyland CaH₂, respectively. NMR spectra were obtained on a Bruker AMX-400.The sites of enzymatic modification of neomycin and gentamicin thatcause drug resistance are shown in FIG. 1 (Daigle, D. M.; et al. Chem.Biol. 1999, 6, 99). Synthesis of biotinylated RNAs and surface plasmonresonance experiments were performed as previously described and K_(d)values were also calculated as previously described (FIG. 2) (Hendrix,M.; et al. J. Am. Chem. Soc. 1997, 119, 3641).

[0053] Antimicrobial Testing: The Kirby-Bauer Disk assay was performedas previously described (Hendrix, M.; et al. J. Am. Chem. Soc. 1997,119, 3641; Phillips, I.; Williams, D. In Laboratory Methods inAntimicrobial Chemotherapy; Gerrod, L., Ed.; Churchill LivingstonePress: Edinburg, 1978; pp 3-30). Reference strains E. coli ATCC 25922and S. aureus ATCC 25923 were obtained as lyophilized pellets (Difco).MIC testing was performed as recommended in the NCCLS Publication M7-A4.

[0054] In vitro translation assays: A coupled transcription-translationassay was performed as previously described with luciferase DNA todetermine the extent of translational inhibition in the presence of thevarious aminoglycosides/mimetics (Greenberg, W. A.; et al. J. Am. Chem.Soc. 1999, 121, 6527). The transcription/translation mixture, or S-30extract, and the reaction buffers were prepared as described previouslywith slight modifications (Greenberg, W. A.; et al. J. Am. Chem. Soc.1999, 121, 6527). The translation assays were performed by mixing all ofthe reagents, various amounts of the compounds to be tested, and the DNAtemplate into a small, RNase-free microcentrifuge tube. The finaladdition was always S-30 extract, and the reaction was maintained at21°+/−1° C. in a water bath. The reaction was terminated after 30minutes by diluting the reaction 10-fold with a luciferase dilutionbuffer containing 1% Triton X-100. Translation yield was determined bymixing 1 μL of the diluted reaction mixture with 50 μL of luciferaseassay reagent (20 mM Tricine, pH 7.8; 15 mM MgSO₄; 0.1 mM EDTA; 33.3 mMDTT; 270 μM coenzyme A; 470 μM luciferin; and 530 μM ATP) and monitoringthe luminescence with a Turner Designs luminometer. For each assay,points were collected in duplicate, and the full assays were performedat least three times.

[0055] 5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine.(Sucheck, S. J.; et al. Angew. Chem., Int. Ed. Engl. 2000, 39, 1080)5-O-Allyl -1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine (Greenberg,W. A.; et al. J. Am. Chem. Soc. 1999, 121, 6527) (264 mg, 0.340 mmol)was dissolved in 14 mL of 1:1 methanol-dichloromethane and was cooled to−78° C. Ozone was bubbled through the solution until it became lightblue in color. The solution was treated with 272 μL of dimethyl sulfideand was allowed to stir one hour while it warmed to room temperature.The solvents were removed under diminished pressure and the crudealdehyde was taken up in 6 mL of 1:1 carbon tetrachloride-acetic acid.The solution was cooled to 0° C. in an ice bath and 305 mg of sodiumchlorite (3.39 mmol) was added in portions over 1 h. The solution waspoured into an ice cold Na₂S₂O₅ solution, acidified to pH 1 with 0.5 NH₂SO₄, extracted with five 50-mL portions ethyl acetate and dried(MgSO₄). The solution was concentrated by co-evaporation with tolueneunder diminished pressure. The product was purified by silica gel flashcolumn chromatography (3×15 cm). Elution with 2:1+1% hexanes-ethylacetate-acetic acid afforded the carboxylic acid as a colorless foam:yield 205 mg (80%); silica gel TLC R_(f) 0.56 (1:1+1% hexanes-ethylacetate-acetic acid); mass spectrum (FAB), m/z 887.1961 (M+Cs)⁺(C₃₅H₃₈N₁₂O₈Cs requires 887.1990).

[0056] General Procedure for the Synthesis of Neamine Dimers.5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine (0.0826mmol/tube) was dissolved in 1.5 mL/tube of dry dichloromethane and wasdistributed into a Quest 210 parallel synthesizer (ArgonautTechnologies; San Carlos, Calif.). To each tube was added 143 mg ofMP-carbodiimide resin (1.15 mmol/g) (Argonaut Technologies; San Carlos,Calif.) followed by the diamine (0.0413 mmol/tube). The solutions wereagitated for 16 hours, filtered and concentrated under diminishedpressure to obtain the dimers as colorless foams.

[0057]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-propylamide,Protected Dimer 4. Yield: 16.9 mg (26%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1569.6495(M+Na)⁺ (C₇₃H₈₂N₂₆Q₁₄Na requires 1569.6401).

[0058]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)butyl-amide,Protected Dimer 5. Yield: 25.9 mg (40%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1583.6550(M+Na)⁺ (C₇₄H₈₄N₂₆O₁₄Na requires 1583.6558).

[0059]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-pentylamide,Protected Dimer 6. Yield: 33.1 mg (51%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1597.6744(M+Na)⁺ (C₇₅H₈₆N₂₆O₁₄Na requires 1597.6714).

[0060]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-hexylamide,Protected Dimer 7. Yield: 25.8 mg (39%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1611.6886(M+Na)⁺ (C₇₆H₈₈N₂₆O₁₄Na requires 1611.6871).

[0061]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-heptylamide,Protected Dimer 8. Yield: 39.6 mg (60%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1625.7021(M+Na)⁺ (C₇₇H₉₀N₂₆O₁₄Na requires 1625.7027).

[0062]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-octylamide,Protected Dimer 9. Yield: 31.7 mg (47%); silica gel TLC R_(f) 0.54 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1639.7137(M+Na)⁺ (C₇₈H₁₂N₂₆O₁₄Na requires 1639.7184).

[0063]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-nonylamide,Protected Dimer 10. Yield: 29.4 mg (44%); silica gel TLC R_(f) 0.59 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1653.7423(M+Na)⁺ (C₇₉H₉₄N₂₆O₁₄Na requires 1653.7340).

[0064]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)decyl-amide,Protected Dimer11. Yield: 36.7 mg (54%); silica gel TLC R_(f) 0.63 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1667.7483(M+Na)⁺ (C₈₀H₉₆N₂₆O₁₄Na requires 1667.7497).

[0065]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-dodecylamide,Protected Dimer 12. Yield: 34.2 mg (50%); silica gel TLC R_(f) 0.65 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1695.7802(M+Na)⁺ (C₈₂H₁₀₀N₂₆O₁₄Na requires 1695.7810).

[0066]N,N′-1,3-bis(5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)-4,7,10-trioxotetradecylamide,Protected Dimer 13. Yield: 32.5 mg (47%); silica gel TLC R_(f) 0.26 (1:1hexanes-ethyl acetate); mass spectrum (MALDI-FTMS), m/z 1715.7432(M+Na)⁺ (C₈₀H₉₆N₂₆O₁₇Na requires 1715.7344).

[0067] General Procedure for the Azide Reduction of Neamine Dimers. TheN,N′-bis(5-ethyl-carboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine)alkylamideswere dissolved in 1.5 mL/tube of dry THF and were distributed into aQuest 210 parallel synthesizer. To each tube was added 150 μL of waterfollowed by 15 μL of 1 N NaOH solution. To the resulting solutions wereadded 10 equivalents of 1 M trimethylphosphine in THF for each tube. Thesolutions were agitated for 16 hours and 100 mg/tube of MP-TsOH resin(1.32 mmol/g) (Argonaut Technolo-gies; San Carlos, Calif.) was added.The solutions were allowed to agitate for 2 hours and were washed withthree 10-mL portions of methanol. The resin bound amines were releasedfrom the resin by washing the resin with two 5-mL portions of 2 Nammonia in methanol. The solutions were concentrated under diminishedpressure to obtain the amines as light yellow syrups. The amines weresubjected to hydrogenolysis conditions without further characterization.

[0068] General Procedure for the Hydrogenolysis of Neamine Dimers. TheN,N′-bis(5-ethyl-carboxyl-6,3′,4′-tri-O-benzylneamine)alkylamides weredissolved in 1 mL/vial of glacial acetic acid. To each vial was added 50μg of 20% Pd(OH)₂/C (Degussa type) and the solutions were placed under 1atm of H₂. The solutions were stirred for 16 hours and were concentratedunder diminished pressure. The deprotected dimers were purified by flashchromatography on silica gel (1×15 cm). Elution with 8:2:5:4 30%ammonium hydroxide-chloroform-ethanol-butanol afforded the dimers as acolorless glasses. The dimers were resuspended in water and applied toDowex 50WX4-50 H⁺ and washed with 5 mL of water. The dimers were elutedwith 3% ammonium hydroxide to obtain the dimers as colorless foams afterlyophilization.

[0069] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)propylamide (4). Yield: 0.9mg (10%); silica gel TLC R_(f) 0.32 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z821.4355 (M+Na)⁺ (C₃₁H₆₂N₁₀O₁₄Na requires 821.4345).

[0070] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)butylamide (5). Yield: 1.4mg (10%); silica gel TLC R_(f) 0.32 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z835.0000 (M+Na)⁺ (C₃₂H₆₄N₁₀O₁₄Na requires 835.4501).

[0071] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)pentylamide (6). Yield: 0.9mg (5.2%); silica gel TLC R_(f) 0.32 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z849.4668 (M+Na)⁺ (C₃₃H₆₆N₁₀O₁₄Na requires 849.4658).

[0072] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)hexylamide (7). Yield: 1.4mg (10%); silica gel TLC R_(f) 0.37 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z863.4838 (M+Na)⁺ (C₃₄H₆₈N₁₀O₁₄Na requires 863.4814).

[0073] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)heptylamide (8). Yield: 1.7mg (8.1%); silica gel TLC R_(f) 0.37 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z855.5173 (M+H)⁺ (C₃₅H₇₁N₁₀O₁₄ requires 855.5151).

[0074] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)octylamide (9). Yield: 4.4mg (26%); silica gel TLC R_(f) 0.58 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z891.5131 (M+Na)⁺ (C₃₆H₇₂N₁₀O₁₄Na requires 891.5127).

[0075] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)nonylamide (10). Yield: 2.4mg (15%); silica gel TLC R_(f) 0.74 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z883.5472 (M+H)⁺ (C₃₇H₇₅N₁₀O₁₄ requires 883.5464).

[0076] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)decylamide (11). Yield: 13mg (13%); silica gel TLC R_(f) 0.74 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z897.5583 (M+H)⁺ (C₃₈H₇₇N₁₀O₁₄ requires 897.5621).

[0077] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)dodecylamide (12). Yield:0.7 mg (3.7%); silica gel TLC R_(f) 0.79 (8:2:5:4 30% ammoniumhydroxide-chloroform-ethanol-butanol); mass spectrum (MALDI-FTMS), m/z947.5729 (M+Na)⁺ (C₄₀H₈₀N₁₀O₁₄Na requires 947.5753).

[0078] N,N′-1,3-bis(5-Ethylcarboxyl-neamine)4,7,10-trioxotetradecylamide(13). Yield: 2.2 mg (12%); silica gel TLC R_(f) 0.79 (8:2:5:4 30%ammonium hydroxide-chloroform-ethanol-butanol); mass spectrum(MALDI-FTMS), m/z 1715.7432 (M+Na)⁺ (C₃₈H₇₆N₁₀O₁₇Na requires 1715.7344).

[0079] Protected Neamine Dimer 14. For a schematic Ugi reaction, seeFIG. 3. In a representative example,5-Ethylcarboxyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzylneamine neamine(60 μg, 79 μmol), methyl isocyanoacetate (36 μL, 397 μmol), benzaldehyde(8 μL, 79 μmol), and diaminododecane (8 mg, 40 μmol) were dissolved in amixture of anhydrous CH₂Cl₂/methanol (1:1, 800 μl). After stirring 48hours at ambient temperature, the reaction was diluted with ethylacetate (5 ml). It was then washed with 1 M HCl (2×5 ml), saturatedsodium bicarbonate (2×5 ml), and brine (1×5 ml). The aqueous extractswere re-extracted with ethyl acetate (2×5 ml). The combined organicextracts were dried (Na₂SO₄), filtered, and concentrated. Flashchromatography (silica gel, gradient hexane to 1:1 hexane/ethyl acetate)yielded protected neamine dimer 14 (21.7 mg, 26%). HRMS (FAB) calcd forC₁₀₄H₁₂₂N₂₈O₂₀ (M+Cs)⁺ 2215.8445, found 2215.8587.

[0080] Neamine Dimer 14. Protected neamine dimer 14 (21.4 mg, 10 μmol)was suspended in ethanol (250 μl). Anhydrous hydrazine (3.2 μl, 100μmol) was added, followed by Raney nickel (˜10 mg) that had been washedthoroughly with ethanol. The reaction was stirred overnight at ambienttemperature, then filtered through a plug of Celite and concentrated.The resulting residue was dissolved in H₂O/AcOH (1:1, 0.04 M). Pd(OH)₂/C(˜10 mg, Degussa type) was added and the reaction stirred under a H₂atmosphere (balloon) overnight. The reaction was filtered through a plugof Celite and lyophilized. Purification was accomplished on CG-50 cationexchange resin, eluting with a gradient of 0 to 40% NH₃/H₂O, to giveneamine dimer 14 (3.4 mg, 26%). ¹H NMR (500 MHz, D₂O) δ7.48-7.36 (10H,bs), 5.71 (2H, d, J=4 Hz), 4.34 (1H, d, J=16 Hz), 4.15 (1H, d, J=15 Hz),3.95-3.32 (32H, m), 2.42-2.35 (2H, m), 1.76 (2H, dd, J=26, 13 Hz),1.27-0.92 (20H, m); ES-MS (neg) calcd for C₆₀H₉₈N₁₂O₂₀ (M−H)⁻ 1306,found 1306.

[0081] Epoxide 15. To 500 mg of perbenzyl-perazido-neamine (Greenberg,W. A.; et al. J. Am. Chem. Soc. 1999, 121, 6527) (0.720 mmol) dissolvedin 5 mL of THF was added 31.7 mg of 60% sodium hydride in paraffin(0.793 mmol). Freshly prepared (S)-glycidol triflate (121 mg, 0.793mmol) was added and the solution stirred overnight at room temperature.The solution was quench with saturated NH₄Cl and partitioned with three50 mL-aliquots of ethyl acetate. The solution was dried (MgSO₄) andconcentrated under diminished pressure. The crude epoxide was purifiedby flash chromatography on silica gel (30×150 mm). The pure product waseluted with 6:1 hexanes-ethyl acetate to afford the epoxide 15 as acolorless foam: yield 405 mg (75%); TLC R_(f) 0.32 (6:1 hexanes-ethylacetate); mass spectrum (MALDIFTMS): m/z 775.3038 [M+Na^(+] (C)₃₆H₄₀N₁₂O₇Na requires 775.3041).

[0082] Epoxide 16. To 280 mg of perbenzyl-perazido-neamine (Greenberg,W. A.; et al. J. Am. Chem. Soc. 1999, 121, 6527) (0.403 mmol) dissolvedin 5 mL of THF was added 17.8 mg of 60% sodium hydride in paraffin(0.444 mmol). Freshly prepared (R)-glycidol triflate (65.3 mg, 0.444mmol) was added and the solution stirred overnight at room temperature.The solution was quench with saturated NH₄Cl and partitioned with three50 mL-aliquots of ethyl acetate. The solution was dried (MgSO₄) andconcentrated under diminished pressure. The crude epoxide was purifiedby flash chromatography on silica gel (30×150 mm). The pure product waseluted with 6:1 hexanes-ethyl acetate to afford the epoxide 16 as acolorless foam: yield: 280 mg (92%); TLC R_(f) 0.32 (6:1 hexanes-ethylacetate); mass spectrum (ESI): m/z 775 [M+Na⁺] (C₃₆H₄₀N₁₂O₇Na requires775).

[0083] Protected Monomer 17. yield: 50.6 mg (65%), TLC R_(f) 0.31(2:2:96 triethylamine-methanol-dichloromethane); mass spectrum(MALDIFTMS): m/z 784.3616 [M+H⁺] (C₃₇H₄₆N₁₃O₇ requires 784.3643).

[0084] Protected Monomer 18. yield: 52.0 mg (66.7%); TLC R_(f) 0.31(2:2:96 triethylamine-methanol-dichloromethane); (MALDIFTMS), m/z784.3632 [M+H⁺] (C₃₇H₄₆N₁₃O₇ requires 784.3643). Protected Dimer 19.yield: 33.2 mg (72%), TLC R_(f) 0.38 (2:2:96triethylamine-methanol-dichloromethane); mass spectrum (MALDIFTMS): m/z1536.6846 [M+H⁺] (C₇₃H₈₆N₂₅O₁₄ requires 1536.6786).

[0085] Protected Dimer 20. yield: 37.5 mg (73.4%); TLC R 0.38 (2:2:96triethylamine-methanol-dichloro-methane); (MALDIFTMS), m/z 1536.6711[M+H⁺] (C₇₃H₈₆N₂₅O₁₄ requires 1536.6785).

[0086] Protected Dimer 21. yield: 39.2 mg (74%), TLC R_(f) 0.38 (2:2:96triethylamine-methanol-dichloromethane); mass spectrum (MALDIFTMS): m/z1593.7404 [M+H⁺] (C₇₆H₁₃N₂₆O₁₄ requires 1593.7365).

[0087] Protected Dimer 22. yield: 29.3 mg (54.9%), TLC R_(f) 0.38(2:2:96 triethylamine-methanol-dichloromethane); (MALDIFTMS): m/z1607.7503 [M+H⁺] (C₇₇H₉₅N₂₆O₁₄ requires 1607.7521).

[0088] Protected Dimer 23. yield: 29.2 mg (54%), TLC R_(f) 0.38 (2:2:96triethylamine-methanol-dichloromethane); mass spectrum (MALDIFTMS): m/z1621.7658 [M+H⁺] (C₇₈H₇₆N₂₆O₁₄ requires 1621.7678).

[0089] Protected Dimer 24. yield: 31.2 mg (58%), TLC R_(f) 0.38 (2:2:96triethylamine-methanol-dichloromethane); (MALDIFTMS): m/z 1637.7633[M+H⁺] (C₇₈H₉₇N₂₆O₁₅ requires 1637.7627).

[0090] Protected Dimer 25. yield: 42.9 mg (90%); TLC R_(f) 0.30 (2:2:96triethylamine-methanol-dichloromethane); (MALDIFTMS), m/z 1593.7299[M+H⁺] (C₇₆H₉₃N₂₆O₁₄ requires 1593.7365).

[0091] Protected Dimer 26. yield: 37.5 mg (77.9%); TLC R_(f) 0.26(2:2:96 triethylamine-methanol-dichloromethane); (MALDIFTMS), m/z1607.7531 [M+H⁺] (C₇₇H₉₅N₂₆O₁₄ requires 1607.7521).

[0092] Protected Dimer 27. yield: 8.2 mg (17.0%), TLC R_(f) 0.23 (2:2:96triethylamine-methanol-dichloromethane); (MALDIFTMS), m/z 1621.7526[M+H⁺] (C₇₈H₉₇N₂₆O₁₄ requires 1621.7677).

[0093] Protected Dimer 28. yield: 8.2 mg (16.7%); TLC R_(f) 0.29 (2:2:96triethylamine-methanol-dichloromethane); mass spectrum (MALDIFTMS): m/z1637.7633 [M+H⁺] (C₇₈H₁₇N₂₆O₁₅ requires 1637.7627).

[0094] Monomer 17. yield: 4.5 mg (34%); TLC R_(f) 0.29; (8:2:5:4ammonium hydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z410.2596 [M+H⁺] (C₁₆H₃₆N₅O₇ requires 410.2609).

[0095] Monomer 18. yield: 14.8 mg (47%); TLC R_(f) 0.27; (8:2:5:4ammonium hydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z432.2421 [M+Na⁺] (C₁₆H₃₅N₅O₇Na requires 432.2429).

[0096] Dimer 19. yield: 6.3 mg (35%); TLC R_(f) 0.21; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 788.4761 [M+H⁺](C₃₁H₆₆N₉O₁₄Na requires 788.4729).

[0097] Dimer 20. yield: 4.9 mg (26%); TLC R_(f) 0.24; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 810.4514 [M+Na⁺](C₃₁H₆₅N₉O₁₄Na requires 810.4543).

[0098] Dimer 21. yield: 5.9 mg (57%); TLC R_(f) 0.27; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (ESI), m/z 843 [M−H⁻](C₃₄H₇₅N₁₀O₁₄ requires 843).

[0099] Dimer 22. yield: 8.1 mg (99%); TLC R_(f) 0.29; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (ESI), m/z 859 [M+H⁺](C₃₅H₇₅N₁₀O₁₄ requires 859).

[0100] Dimer 23. yield: 2.6 mg (16%); TLC R_(f) 0.27; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 895.5439 [M+Na⁺](C₃₆H₇₆N₁₀O₁₄ requires 895.5435).

[0101] Dimer 24. yield: 5.2 mg (41%); TLC R_(f) 0.28; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 889.5571 [M+H⁺](C₃₆H₇₇N₁₀O₁₅ requires 889.5564).

[0102] Dimer 25. yield: 6.0 mg (51%); TLC R_(f) 0.26; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (ESI), m/z 843 [M−H⁻](C₃₄H₇₁N₁₀O₁₄ requires 843).

[0103] Dimer 26. yield: 3.1 mg (28%); TLC R_(f) 0.29; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (ESI), m/z 859 [M+H⁺](C₃₅H₇₅N₁₀O₁₄ requires 859).

[0104] Dimer 27. yield: 13.0 mg (90%); TLC R_(f) 0.27; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 895.5439 [M+Na⁺](C₃₆H₇₆N₁₀O₁₄ requires 895.5435).

[0105] Dimer 28. yield: 8.1mg (64%); TLC R_(f) 0.27; (8:2:5:4 ammoniumhydroxide-chloroform-ethanol-butanol); (MALDIFTMS), m/z 889.5565 [M+H⁺](C₃₆H₇₇N₁₀O₁₅ requires 889.5564).

What is claimed is:
 1. A bifunctional antibiotic comprising a first anda second pharmacophore and a linkage for linking said first and saidsecond pharmacophores, each of said first and second pharmacophoreshaving a binding affinity for the A-site of bacterial 16S rRNAsufficient to inhibit translation at clinically effectiveconcentrations, said first and second pharmacophores being identical toone another or different from one another, said linkage having a lengthand structure for enabling said first and second pharmacophores to bindsimultaneously to a single A-site of bacterial 16S rRNA.
 2. Abifunctional antibiotic according to claim 1 wherein at least one ofsaid first and second pharmacophores is inhibitory of APH(2″) activitywith respect to bifunctional enzyme AAC(6′)-APH(2″), the inhibitoryactivity being sufficient, at clinically effective concentrations, todiminish deactivation of said bifunctional antibiotic by saidbifunctional enzyme AAC(6′)-APH(2″).
 3. A bifunctional antibioticaccording to claim 1 represented by the following structure: wherein:

Y¹ and Y² are the first and second pharmacophore respectively and areboth represented by:

R¹ and R² are each independently selected from the group of radicalsconsisting of —H and —CH(Ph)CONHCH₂CO₂H; and X is the linkage and isselected from the group of diradicals consisting of —(CH₂)_(n)— and—[(CH₂)₂O(CH₂)₃]₂O, where 3≦n≧12.
 4. A bifunctional antibiotic accordingto claim 1 represented by the following structure:

wherein Y¹ and Y² are the first and second pharmacophore respectivelyand are both represented by:

the stereochemistry is either (S,S) or (R,R).
 5. A bifunctionalantibiotic according to claim 4 represented by the following structure:


6. A bifunctional antibiotic according to claim 4 represented by thefollowing structure:


7. A bifunctional antibiotic according to claim 1 represented by thefollowing structure:

wherein: Y¹ and Y² are the first and second pharmacophore respectivelyand are both represented by:

X is the linkage and is selected from the group of diradicals consistingof —(CH₂)_(n)— and —[(CH₂)₂]₂O, where 2≦n≦4; and the stereochemistry iseither (S,S) or (R,R).
 8. A bifunctional antibiotic according to claim 7represented by the following structure:


9. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


10. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


11. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


12. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


13. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


14. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


15. A bifunctional antibiotic according to claim 7 represented by thefollowing structure:


16. A bifunctional antibiotic according to claim 1 wherein the first andsecond pharmacophore are independently selected from the groupconsisting of neamine, neomycin B, and gentamincin C₁.
 17. A process forinhibiting translation within a bacterium having 16S rRNA with anA-site, said process comprising the step of contacting the bacteriumwith a concentration of a bifunctional antibiotic selected from claims1-16 sufficient to inhibit translation.
 18. A process for simultaneouslyinhibiting translation and APH(2″) activity within a bacterium havingboth 16S rRNA with an A-site and the bifunctional enzymeAAC(6′)-APH(2″), said process comprising the step of contacting thebacterium with a concentration of a bifunctional antibiotic selectedfrom claims 1-16 sufficient to inhibit translation and APH(2″) activity.